The transcription factor GATA-1 is essential for normal erythroid development. Study of GATA-1-mouse embryonic stem (ES) cells previously showed that adult (definitive) erythroid precursors arrest at the proerythroblast stage and undergo apoptosis. How GATA-1 functions to control terminal erythroid maturation is unknown. Although various domains of the protein have been defined in heterologous cell transfection systems or in vitro, those relevant to function in an erythroid environment are uncertain, largely due to the absence of a suitable assay for GATA-1 function. In an effort to address these aspects, a novel erythroid cell line (G1E) was generated directly from in vitro differentiation GATA-1-ES cells. This proerythroblast-like cell line lacks GATA-1, but retains the ability to complete terminal erythroid maturation upon reintroduction of functional GATA-1 cDNA by retroviral infection. Using this stringent assay, a structure-function analysis of GATA-1, and its related family members (GATA-2 and GATA-3), will be performed. Particular attention will be directed to comparing the abilities of these factors (GATA-1, 2, 3) to trigger terminal erythroid maturation, the role (if any) of amino- and carboxyl-terminal activation domains for in vivo GATA-1 function, and the role and specificity of the DNA-binding domain for function in an erythroid cell environment. With G1E cells harboring conditionally active GATA-factors (GATA-estrogen receptor fusions), a systematic search for RNAs induced (or repressed) under the direct control of these factors during terminal erythroid maturation will be performed. These "difference" transcripts will ultimately provide the reagents with which to understand why deficiency of GATA-1 leads to developmental arrest. Finally, the functional redundancy of GATA-1 and GATA-2 in the early phase of erythroid development, which is suggested by the phenotype of GATA-1-erythroid precursors, will be tested formally by the generation of targeted ES cells lacking both GATA-1 and GATA-2 function. Ultimately, these studies will reveal how GATA-1, and other GATA-factors, function within erythroid cells to program their differentiation.